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Frequent epigenetic inactivation of cystatin M in breast carcinoma

Abstract

Cystatin M is a potent endogenous inhibitor of lysosomal cysteine proteases. In breast carcinoma, cystatin M expression is frequently downregulated. It has been shown that cystatin M expression suppressed growth and migration of breast cancer cells. We examined the methylation status of the CpG island promoter of cystatin M in four breast cancer cell lines (MDAMB231, ZR75-1, MCF7 and T47D), in 40 primary breast carcinoma and in corresponding normal tissue probes by combined bisulphite restriction analysis. To investigate the effects of cystatin M expression on the growth of breast carcinoma, cystatin M was transfected in T47D. The cystatin M promoter was highly methylated in all four-breast cancer cell lines. Primary breast tumours were significantly more frequently methylated compared to normal tissue samples (60 vs 25%; P=0.006 Fisher's exact test). Treatment of breast cancer cells with 5-aza-2′-deoxycytidine (5-Aza-CdR), reactivated the transcription of cystatin M. Transfection of breast carcinoma cells with cystatin M caused a 30% decrease in colony formation compared to control transfection (P=0.002). Our results show that cystatin M is frequently epigenetically inactivated during breast carcinogenesis and cystatin M expression suppresses the growth of breast carcinoma. These data suggest that cystatin M may encode a novel epigenetically inactivated candidate tumour suppressor gene.

Introduction

Cysteine proteases are known to play a crucial role in cancer diseases (Krepela, 2001). Cysteine proteases can be involved in enhanced cell survival and proliferation, escape from immune surveillance, cell adhesion and migration, remodelling and invasion of the extracellular matrix (Nomura and Katunuma, 2005). Cystatins are potent inhibitors of endogenous mammalian lysosomal cysteine proteases and protect cells against uncontrolled proteolysis (Abrahamson et al., 2003). Disturbance of the balance between proteases and their cystatins can lead to irreversible damage and tumour development (Calkins and Sloane, 1995; Henskens et al., 1996). Cystatin M is a new member of the human cystatin gene family and demonstrates more diverse tissue distribution and target specificity than other cystatins (Ni et al., 1997; Sotiropoulou et al., 1997). Cystatin M was first identified and cloned by differential RNA display as a transcript that is downregulated in metastatic breast cancer cells when compared to primary breast cancer cells (Ni et al., 1997; Sotiropoulou et al., 1997). Cystatin M is a low molecular mass protein sharing 27–32% homology to other cystatins. The protein can be secreted from cells in a glycosylated (17 kDa) and unglycosylated (14.4 kDa) form (Ni et al., 1997). It has been reported that constitutive expression of cystatin M in human breast cancer cells MDA-MB-435S significantly reduces in vitro cell proliferation, migration, Matrigel invasion and tumour–endothelial cell adhesion (Shridhar et al., 2004). Moreover, the ectopic expression of cystatin M in scid mice strongly delayed tumour growth and lowered the rate of metastases in lung and liver (Zhang et al., 2004).

Cystatin M has been assigned to chromosome region 11q13, which is the site of loss of heterozygosity (LOH) in several cancer types and is believed to harbour tumour suppressor genes (Stenman et al., 1997; Srivatsan et al., 2002). Cystatin M regulates the activity of cathepsin B and cathepsin L, which are the two most important cysteine proteases implicated in tumour cell invasion and metastasis (Lah and Kos, 1998; Frosch et al., 1999). Cystatin M is consistently expressed in normal and premalignant breast epithelial cells, but downregulated in malignant breast cancer cell lines (Sotiropoulou et al., 1997). However the mechanisms of cystatin M downregulation has not been elucidated.

Epigenetic inactivation of tumour suppressor genes plays a fundamental role in the development and progression of cancer (Jones and Baylin, 2002). To determine if epigenetic silencing of cystatin M occurs in pathogenesis of breast cancer, we investigated the methylation status of the cystatin M promoter in primary breast carcinoma. Moreover, we transfected cystatin M into breast cancer cells and analysed colony formation.

Results

Hypermethylation of the cystatin M CpG island in breast cancer

It has been reported that cystatin M expression is significantly reduced in breast tumours (Sotiropoulou et al., 1997) and that its expression inhibits growth of breast cancer cells (Shridhar et al., 2004). Epigenetic inactivation of tumour suppressor genes by hypermethylation of their CpG island promoters plays an essential role in tumour pathogenesis (Herman and Baylin, 2003). Therefore, we investigated whether aberrant methylation of the cystatin M promoter occurred in breast cancer. The promoter region of cystatin M was screened for the presence of CpG dinucleotides by in silicio analysis (Figure 1a). A 500 bp long CpG island covering the transcription initiation site and the first exon was revealed. To elucidate methylation of cystatin M promoter in breast cancer, we performed combined bisulphite restriction analysis (COBRA) assays (Xiong and Laird, 1997). First, we analysed the methylation status of the cystatin M promoter in four breast carcinoma cell lines (ZR75-1, MCF7, T47D and MDAMB231; Figure 1b). ZR75-1 and MCF7 were completely methylated and MDAMB231 and T47D were partially methylated (Figure 1b). We confirm the methylation status of the cystatin M promoter by sequencing polymerase chain reaction (PCR) products obtained from breast cancer cell lines ZR75-1 and T47D, and from a normal breast tissue sample (Figure 2). Subsequently, we analysed methylation of the cystatin M CpG island promoter in primary breast carcinomas and corresponding normal tissue samples (Figure 1c). In 24 out of 40 (60%) breast carcinomas, the promoter of cystatin M was methylated. In contrast, in only seven out of 28 (25%) normal breast tissue samples cystatin M was methylated (Table 1). However, methylation was lower than in the corresponding tumour cases. Hypermethylation was significantly more frequent in tumours compared to normal samples (P=0.006, Fisher's exact test). Methylation of cystatin M was compared with different clinicopathological data. No significant correlation with tumour entities, grading and age of onset was found (Table 1). It was observed that oestrogen receptor-positive tumours were more frequently methylated than oestrogen receptor-negative tumours. This tendency was not significant for cystatin M (P=0.061), but was significant for Ras association domain family 1A (RASSF1A) (P=0.009).

Figure 1
figure1

Methylation analysis of the cystatin M CpG island in breast carcinoma. (a) Map of the cystatin M promoter region. The first exon is indicated and the arrow marks the transcription initiation site. The CpG island was determined by CpGplot (www.ebi.ac.uk/emboss/cpgplot) with default parameters. CpGs and Taq-I sites after in silicio bisulphite treatment are indicated. Arrowheads mark positions of primers (CystMU1, CystMU2 and CystML1) utilized for COBRA. (b) COBRA of breast cancer cell lines. PCR products (287 bp) from bisulphite-treated DNA obtained from different breast cancer cell lines were digested (+) and mock digested (−) with Taq-I. In vitro methylated DNA (Methyl) was utilized as positive control. (c) COBRA of primary breast cancer samples and matching normal tissue. PCR products from bisulphite-treated DNA obtained from primary tumours (T) and normal samples (N) were digested (+) and mock-digested (−) with Taq-I. The sizes of a 100 bp ladder (M) are indicated.

Figure 2
figure2

Bisulphite sequencing of the cystatin M promoter in breast cancer. Bisulphite-treated DNA isolated from ZR75-1, T47D and the normal breast sample N4720 was amplified and cloned. For technical reasons, the complementary strand was sequenced. Positions of CpGs are underlined.

Table 1 Cysatin M methylation and RASSF1A methylation in breast cancer and matching normal tissues

The methylation status of the RASSF1A tumour suppressor gene for the analysed primary breast cancer samples was available from the previous work (Dammann et al., 2001) (Table 1). We observed a significant association between methylation of cystatin M and RASSF1A. Primary breast cancer cases with methylated cystatin M had a significantly increased frequency of RASSF1A methylation (79%) compared to cases with unmethylated cystatin M (38%, P=0.018 Fisher's exact test).

Inhibition of DNA methylation reactivates cystatin M expression

To correlate the methylation status with cystatin M transcription, we performed reverse trancriptase–polymerase chain reaction (RT–PCR) in breast cancer cell lines T47D and ZR75-1, and in normal human mammary epithelial cells (HMEC) (Figure 3). High level of cystatin M transcript was detected in HMEC. In T47D, which harbours a partially methylated promoter, very low level of cystatin M was found, and in the completely methylated ZR75-1 cells, no cystatin M was observed (Figure 3). Subsequently, we treated these cancer cell lines with two concentrations (5 and 10 μ M) of 5-Aza-CdR, a drug that inhibits DNA methylation (Jones and Taylor, 1980). After 4 days of treatment, we detected an increase of cystatin M transcripts in breast cancer cell lines T47D and ZR75-1 (Figure 3).

Figure 3
figure3

Expression of cystatin M. Cystatin M expression was analysed by RT–PCR in breast cancer cell lines T47D, ZR75-1 and in normal human mammary epithelial cells (HMEC). The cells were treated for 4 days with indicated concentrations of 5-Aza-CdR and the isolated RNA was amplified by RT–PCR. PCR products of cystatin M (223 bp) were resolved on a 2% tris-borate-EDTA (TBE) agarose gel. Expression of GAPDH was determined as a control for RNA integrity. A 100 bp ladder (M) is indicated.

Transfection of cystatin M inhibits colony formation

Previously, it has been shown that cystatin M expression inhibits growth of the breast cancer cell line MDA-MB-435SD (Shridhar et al., 2004). To confirm function of cystatin M as a tumour suppressor gene in breast carcinogenesis, we transfected the breast cancer cell line T47D with cystatin M and with a vector control (Figure 4). After selection with G418 for 4 weeks, we observed a 30% decrease in colony formation in cells transfected with cystatin M (n=46±3) in comparison to T47D cells transfected with a vector control (n=65±4) (P=0.002; one-way analysis of variance (ANOVA) test).

Figure 4
figure4

Colony formation assay for breast cancer cell line T47D. (a) After transfection with cystatin M and the empty vector (pcDNA3.1), cells were grown in selective medium for four weeks and stained by Giemsa. (b) Numbers of colonies were evaluated and plotted. Means and s.d. of three independent experiments are indicated. The difference between cystatin M and control transformations was significant (P=0.002; one-way ANOVA test).

Discussion

Cystatin M, a potent inhibitor of human lysosomal proteases, was identified as a gene frequently transcriptionally downregulated during progression of breast cancer (Ni et al., 1997; Sotiropoulou et al., 1997; Zhang et al., 2004). In our study, we demonstrated that hypermethylation of the cystatin M promoter occurs frequently in primary breast carcinomas and breast cancer cell lines. This result suggests that aberrant methylation of the cystatin M CpG island is an important mechanism of its silencing. To our knowledge, this is the first demonstration of epigenetic silencing of cystatin M in cancer. Complete loss of expression of cystatin M was previously demonstrated in different metastatic tumour cell lines (Sotiropoulou et al., 1997). Here we show that in several breast cancer cell lines cystatin M is highly reduced and was re-expressed after inhibition of DNA methyltransferase with 5-Aza-CdR. These data confirm that hypermethylation of the cystatin M promoter inhibits its transcription. It has been suggested that cystatin M inactivation is associated with progression of a primary tumour to a metastatic phenotype (Sotiropoulou et al., 1997). Subsequently, it has been demonstrated that constitutive expression of cystatin M in human breast carcinoma MDA-MB-435S cells reduces in vitro cell proliferation, migration, Matrigel invasion and adhesion to endothelial cells (Shridhar et al., 2004). Our data support this result as re-expression of cystatin M in the breast cancer cells T47D significantly reduced colony formation of these cells. In vivo experiments with scid mice transplanted with breast cancer cells expressing cystatin M showed significantly delayed breast tumour growth and lowered metastatic burden in the lung and liver at secondary sites (Zhang et al., 2004). Thus, it has been proposed that cystatin M may function as candidate tumour suppressor for breast carcinogenesis (Zhang et al., 2004).

Cystatin M is a potent inhibitor of cysteine peptidases, including papain, cathepsin B, cathepsin V and cathepsin L (Ni et al., 1997; Cheng et al., 2006). These peptidases play key roles in intracellular protein degradation. Cathepsins B and L promote tumour growth, invasion and metastasis through degradation of extracellular connective matrices and through endothelial cell growth-directed activities (Krepela, 2001). Thus, deregulation of cystatin M in tumours may enhance tumorigenic and metastatic function of cathepsins. Re-expression of cystatin M in breast cancer cells reduced the level of the potent mitogenic and angiogenic factor autotaxin (Song et al., 2006). Additionally, cystatin M has important regulatory functions in human epidermal differentiation (Zeeuwen, 2004). In wound healing, cystatin M expression was not found in the edge of migrating keratinocytes and in epidermal neoplasia cystatin M was only detected in differentiated cells and keratinized cell nests (Zeeuwen et al., 2002). Cystatin M downregulation was also observed in different cancer cells, including prostate cancer, colon cancer, glioblastoma, lung cancer and melanoma (Sotiropoulou et al., 1997; Shridhar et al., 2004). This indicates that cystatin M may be epigenetically downregulated in other cancers. In contrast, in oropharyngeal and skin squamous cell carcinoma, an upregulation of cystatin M expression was reported (Vigneswaran et al., 2003; Haider et al., 2006). Further studies should clarify if aberrant methylation (hyper- or hypomethylation) of cystatin M is a frequent process in pathogenesis of different cancer entities.

In our previous work, we have identified a novel Ras effector homologue termed RASSF1A (Dammann et al., 2000). It has been demonstrated that RASSF1A is frequently downregulated in breast cancer and this silencing was associated with aberrant methylation of the RASSF1A CpG island promoter (Burbee et al., 2001; Dammann et al., 2001). Comparison of methylation of cystatin M with the tumour suppressor gene RASSF1A showed a significant correlation. Previously, we have shown that progressive RASSF1A hypermethylation was found during senescence of normal human mammary epithelial cells (Strunnikova et al., 2005). Methylation of matching normal tissue of breast cancer patients may be related to methylation reported in ageing or attributed to epigenetic field defect and infiltrating tumour cells (Ahuja and Issa, 2000; Waki et al., 2003; Guo et al., 2004).

In summary, our results indicate that cystatin M may function as an epigenetically silenced tumour suppressor in breast carcinogenesis. These results support the suggestion that cystatin M inactivation is a characteristic process in the progression of breast cancer and may play an important role in the safeguarding against this cancer type. The exact role of cystatin M inactivation combined with overexpression of cysteine proteases and the consequential proteolytic imbalance remains to be elucidated.

Materials and methods

Tissues and cell lines

In our study, we analysed 40 primary breast carcinoma and 28 corresponding normal breast tissues. All tissues were classified and obtained from the Pathology Department of the City of Hope National Medical Center (Duarte, CA, USA) and were described previously (Dammann et al., 2001). Four human breast cancer cell lines ZR75-1, T47D, MDAMB231 and MCF7 were obtained from the American Type Culture Collection and were cultured in the recommended growth medium. RNA was isolated and RT–PCR was performed as described below. Genomic DNA was extracted from frozen tissues and cultured cells by a standard phenol/chloroform procedure.

Bisulphite treatment of DNA and methylation analysis

Methylation of the cystatin M promoter region was determined by bisulphite modification of genomic DNA (Clark et al., 1994; Dammann et al., 2000). Combined bisulphite restriction analysis (COBRA) was performed as described previously (Xiong and Laird, 1997). Briefly, 100 ng of bisulphite-treated DNA were amplified with primers CystMU1: 5′-IndexTermTTGTATTGGTATTTGTTGTTGGGGATTG and CystML1: 5′-IndexTermCAAAATACCACCAAAACCAAACCCAAC. A hot-started PCR in 25 μl reaction buffer containing 0.2 mM dNTP mix, 1.5 mM MgCl2, 10 pmol of each primer and Taq polymerase (InViTek GmBH, Berlin, Germany) at 92°C for 30 s, 60°C for 30 s, and 72°C for 30 s for 20 cycles was performed. A seminested PCR was carried out using an internal primer CystMU2: 5′-IndexTermGGGTTAGGTGTGTTTTGGAGGGTAGG and primer CystML1 with similar conditions as described above, but for 30 cycles. Expected product size after seminested PCR was 287 bp. For restriction analysis, PCR products were digested with 10 U of Taq-I (New England Biolabs, Beverly, MA, USA) according to conditions specified by the manufacturer and analysed on a 2% Tris-borate ethylenediamine tetra acetic acid (EDTA) agarose gel.

5-Aza-CdR treatment and RT–PCR analysis

Two breast cancer cell lines (ZR75-1 and T47D) and normal breast epithelial cells (HMEC) were treated with 5-Aza-CdR) (Sigma, Taufkirchen, Germany). Briefly, 2 × 106 cells were grown for 4 days in the presence of different concentrations (0, 5 and 10 μ M) of 5-Aza-CdR. RNA was isolated using Trizol-Reagent (Invitrogen, Karlsruhe, Germany). Reverse transcription was performed with poly-dT-primer and 1 μg RNA in 25 μl of RT-mix with avian myeloblastosis virus-reverse transcriptase (AMV-RT) (Promega GmBH, Heidelberg, Germany) for 1 h at 42°C. Subsequently, 2 μl of cDNA was amplificated at 60°C, for 38 cycles using following cystatin M-specific primers: forward primer, 5′-IndexTermGCAGAAGGCGGCGCAGGC and reverse primer, 5′-IndexTermCTCCTGCTGCGCCCCTGCTG with an expected product size of 223 bp. Amplification of the glyceraldehyde-3-phosphate dehydrogenase (GAPDH) transcript was carried out to verify integrity of RNA. PCR products were separated on 2% Tris-borate EDTA agarose gels.

Cystatin M transfection and colony formation assay

To transfect cystatin M into the breast cancer cell line T47D, we cloned cystatin M into the mammalian expression vector pcDNA3.1+ vector (Invitrogen, Carlsbad, CA, USA). We obtained plasmid IMAGP998J016038Q from RZPD (Deutsches Ressourcenzentrum für Genomforschung GmbH, Berlin, Germany), which contains the full-length cDNA clone of cystatin M (509 bp). The cDNA of cystatin M was isolated by restriction with HindIII and EcoRI (New England BioLabs GmbH, Frankfurt am Main, Germany) and ligated into pCDNA3.1+ vector. Subsequently, 0.3 μg of plasmid DNA was transfected into T47D cells using Lipofectamine 2000 (Invitrogen, Carlsbad, CA, USA) for 6 h and cells were selected in Rosewell's Park Memorial Institute (RPMI) (Biochrom AG, Berlin, Germany) with 320 μg/ml G418 (PAA Laboratories GmbH, Pasching, Germany). After 4 weeks, clones were stained with Giemsa and colony formation was evaluated. As a negative control, T47D cells transfected with pCDNA3.1+ were utilized. Colony formation assay was repeated three times and means were determined.

Statistical evaluation

Statistical analysis was carried out using SPSS 12. Categorical variables were plotted into contingency tables and evaluated using the Fisher's exact test. Results of colony formation tests were plotted as mean±s.d. Mean values of colony formations were compared using one-way ANOVA test. All reported P-values are two-sided and considered significant when P was <0.05.

Abbreviations

COBRA:

combined bisulphite restriction analysis

5-Aza-CdR:

5-aza-2′-deoxycytidine

RASSF1A :

Ras association domain family 1A

References

  1. Abrahamson M, Alvarez-Fernandez M, Nathanson CM . (2003). Cystatins. Biochem Soc Symp 70: 179–199.

    CAS  Article  Google Scholar 

  2. Ahuja N, Issa JP . (2000). Aging, methylation and cancer. Histol Histopathol 15: 835–842.

    CAS  PubMed  Google Scholar 

  3. Burbee DG, Forgacs E, Zochbauer-Muller S, Shivakumar L, Fong K, Gao B et al. (2001). Epigenetic inactivation of RASSF1A in lung and breast cancers and malignant phenotype suppression. J Natl Cancer Inst 93: 691–699.

    CAS  Article  Google Scholar 

  4. Calkins CC, Sloane BF . (1995). Mammalian cysteine protease inhibitors: biochemical properties and possible roles in tumor progression. Biol Chem Hoppe Seyler 376: 71–80.

    CAS  PubMed  Google Scholar 

  5. Cheng T, Hitomi K, van Vlijmen-Willems IM, de Jongh GJ, Yamamoto K, Nishi K et al. (2006). Cystatin M/E is a high affinity inhibitor of cathepsin v and cathepsin l by a reactive site that is distinct from the legumain-binding site. J Biol Chem 281: 15893–15899.

    CAS  Article  Google Scholar 

  6. Clark SJ, Harrison J, Paul CL, Frommer M . (1994). High sensitivity mapping of methylated cytosines. Nucleic Acids Res 22: 2990–2997.

    CAS  Article  Google Scholar 

  7. Dammann R, Li C, Yoon JH, Chin PL, Bates S, Pfeifer GP . (2000). Epigenetic inactivation of a RAS association domain family protein from the lung tumour suppressor locus 3p21.3. Nat Genet 25: 315–319.

    CAS  Article  Google Scholar 

  8. Dammann R, Yang G, Pfeifer GP . (2001). Hypermethylation of the cpG island of Ras association domain family 1A (RASSF1A), a putative tumor suppressor gene from the 3p21.3 locus, occurs in a large percentage of human breast cancers. Cancer Res 61: 3105–3109.

    CAS  PubMed  Google Scholar 

  9. Frosch BA, Berquin I, Emmert-Buck MR, Moin K, Sloane BF . (1999). Molecular regulation, membrane association and secretion of tumor cathepsin B. Apmis 107: 28–37.

    CAS  Article  Google Scholar 

  10. Guo M, House MG, Hooker C, Han Y, Heath E, Gabrielson E et al. (2004). Promoter hypermethylation of resected bronchial margins: a field defect of changes? Clin Cancer Res 10: 5131–5136.

    CAS  Article  Google Scholar 

  11. Haider AS, Peters SB, Kaporis H, Cardinale I, Fei J, Ott J et al. (2006). Genomic analysis defines a cancer-specific gene expression signature for human squamous cell carcinoma and distinguishes malignant hyperproliferation from benign hyperplasia. J Invest Dermatol 126: 869–881.

    CAS  Article  Google Scholar 

  12. Henskens YM, Veerman EC, Nieuw Amerongen AV . (1996). Cystatins in health and disease. Biol Chem Hoppe Seyler 377: 71–86.

    CAS  PubMed  Google Scholar 

  13. Herman JG, Baylin SB . (2003). Gene silencing in cancer in association with promoter hypermethylation. N Engl J Med 349: 2042–2054.

    CAS  Article  Google Scholar 

  14. Jones PA, Baylin SB . (2002). The fundamental role of epigenetic events in cancer. Nat Rev Genet 3: 415–428.

    CAS  Article  Google Scholar 

  15. Jones PA, Taylor SM . (1980). Cellular differentiation, cytidine analogs and DNA methylation. Cell 20: 85–93.

    CAS  Article  Google Scholar 

  16. Krepela E . (2001). Cysteine proteinases in tumor cell growth and apoptosis. Neoplasma 48: 332–349.

    CAS  PubMed  Google Scholar 

  17. Lah TT, Kos J . (1998). Cysteine proteinases in cancer progression and their clinical relevance for prognosis. Biol Chem 379: 125–130.

    CAS  PubMed  Google Scholar 

  18. Ni J, Abrahamson M, Zhang M, Fernandez MA, Grubb A, Su J et al. (1997). Cystatin E is a novel human cysteine proteinase inhibitor with structural resemblance to family 2 cystatins. J Biol Chem 272: 10853–10858.

    CAS  Article  Google Scholar 

  19. Nomura T, Katunuma N . (2005). Involvement of cathepsins in the invasion, metastasis and proliferation of cancer cells. J Med Invest 52: 1–9.

    Article  Google Scholar 

  20. Shridhar R, Zhang J, Song J, Booth BA, Kevil CG, Sotiropoulou G et al. (2004). Cystatin M suppresses the malignant phenotype of human MDA-MB-435S cells. Oncogene 23: 2206–2215.

    CAS  Article  Google Scholar 

  21. Song J, Jie C, Polk P, Shridhar R, Clair T, Zhang J et al. (2006). The candidate tumor suppressor CST6 alters the gene expression profile of human breast carcinoma cells: down-regulation of the potent mitogenic, motogenic, and angiogenic factor autotaxin. Biochem Biophys Res Commun 340: 175–182.

    CAS  Article  Google Scholar 

  22. Sotiropoulou G, Anisowicz A, Sager R . (1997). Identification, cloning, and characterization of cystatin M, a novel cysteine proteinase inhibitor, down-regulated in breast cancer. J Biol Chem 272: 903–910.

    CAS  Article  Google Scholar 

  23. Srivatsan ES, Chakrabarti R, Zainabadi K, Pack SD, Benyamini P, Mendonca MS et al. (2002). Localization of deletion to a 300 Kb interval of chromosome 11q13 in cervical cancer. Oncogene 21: 5631–5642.

    CAS  Article  Google Scholar 

  24. Stenman G, Astrom AK, Roijer E, Sotiropoulou G, Zhang M, Sager R . (1997). Assignment of a novel cysteine proteinase inhibitor (CST6) to 11q13 by fluorescence in situ hybridization. Cytogenet Cell Genet 76: 45–46.

    CAS  Article  Google Scholar 

  25. Strunnikova M, Schagdarsurengin U, Kehlen A, Garbe JC, Stampfer MR, Dammann R . (2005). Chromatin inactivation precedes de novo DNA methylation during the progressive epigenetic silencing of the RASSF1A promoter. Mol Cell Biol 25: 3923–3933.

    CAS  Article  Google Scholar 

  26. Vigneswaran N, Wu J, Zacharias W . (2003). Upregulation of cystatin M during the progression of oropharyngeal squamous cell carcinoma from primary tumor to metastasis. Oral Oncol 39: 559–568.

    CAS  Article  Google Scholar 

  27. Waki T, Tamura G, Sato M, Motoyama T . (2003). Age-related methylation of tumor suppressor and tumor-related genes: an analysis of autopsy samples. Oncogene 22: 4128–4133.

    CAS  Article  Google Scholar 

  28. Xiong Z, Laird PW . (1997). COBRA: a sensitive and quantitative DNA methylation assay. Nucleic Acids Res 25: 2532–2534.

    CAS  Article  Google Scholar 

  29. Zeeuwen PL . (2004). Epidermal differentiation: the role of proteases and their inhibitors. Eur J Cell Biol 83: 761–773.

    CAS  Article  Google Scholar 

  30. Zeeuwen PL, van Vlijmen-Willems IM, Egami H, Schalkwijk J . (2002). Cystatin M/E expression in inflammatory and neoplastic skin disorders. Br J Dermatol 147: 87–94.

    CAS  Article  Google Scholar 

  31. Zhang J, Shridhar R, Dai Q, Song J, Barlow SC, Yin L et al. (2004). Cystatin m: a novel candidate tumor suppressor gene for breast cancer. Cancer Res 64: 6957–6964.

    CAS  Article  Google Scholar 

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Acknowledgements

This study was supported by Grants BMBF FKZ01ZZ0104 and DFG DA552-1 to Reinhard Dammann and WR (FKZ13/13) to Undraga Schagdarsurengin and NIH Grant CA88873 to Gerd P Pfeifer.

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Schagdarsurengin, U., Pfeifer, G. & Dammann, R. Frequent epigenetic inactivation of cystatin M in breast carcinoma. Oncogene 26, 3089–3094 (2007). https://doi.org/10.1038/sj.onc.1210107

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Keywords

  • cystatin M
  • tumour suppressor gene
  • epigenetic inactivation
  • methylation
  • breast cancer
  • CST6

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